Objective: To investigate the role of PHP14 in epithelial-mesenchymal transition and its possible mechanism. Methods: A cell model was established to study epithelial-mesenchymal transition in mouse hepatocytes AML-12. Overexpression of PHP14 in AML-12. Morphological observation was carried out by light microscope. The expression of related markers in epithelial-mesenchymal transition of AML-12 was detected by real-time fluorescent quantitative PCR and Western blot. The interaction between PHP14 and Vimentin was detected by immunoprecipitation. Results: Mouse hepatocyte AML-12 can be used as a good cell model to study epithelial-mesenchymal transition. Overexpression of PHP14 in AML-12 can promote epithelial-mesenchymal transition and enhance the motility of AML-12 cells. Overexpression of PHP14 upregulated Vimentin, but had no effect on the expression of other epithelial-mesenchymal transition related markers. Further studies showed that PHP14 could interact with Vimentin. Conclusion: PHP14 can stabilize Vimentin protein level and participate in and regulate epithelial-mesenchymal transition, by interacting with Vimentin.
Objective: To better understand the possible molecular mechanism of the interaction between the host and human respiratory syncytial virus (RSV), the long non-coding RNA expression profile in A549 cells infected by RSV was investigated. Methods: After 24 hours of A549 cells infected with 1 MOI RSV and extracted for RNA samples, lncRNA differential expression profile was screened by Agilent’s lncRNA expression profile chip and compared with the A549 cell control. LncRNA differential expression profile was verified by real-time quantitative PCR (qRT-PCR). Bioinformatics analysis of differential mRNA and lncRNA was performed by GO and KEGG. Results: A total of 1 463 lncRNA and 1 552 mRNAs were significantly up-regulated in RSV-infected A549 cells compared with A549 cell control, while 944 lncRNA and 1 489 mRNAs were down-regulated. LncRNA and the predicted target genes were mainly enriched in the immune response. The expression profile of the selected lncRNA was confirmed by qRT-PCR. Conclusion: LncRNA expression profile changes significantly after RSV infection of A549 cells, but the underlining mechanism in the interaction between RSV and host needs to be further explored.
It is well known that the fatality rate of rabies, an acute zoonotic disease, is nearly 100%. As one of the high incidence areas in central China, investigating rabies epidemic in Hubei province not only can understand the current risk of Chinese rabies epidemic, but also can provide effective suggestions for local and national rabies prevention and control. Firstly, descriptive analysis turned out that the rabies cases of Hubei province are decreasing, but the exposed cases of rabies are increasing. Especially, rabies cases in the central and western regions of Hubei are slightly greater than the east, while rabies cases for men is significantly greater than for women. Moreover, the rabies cases of middle-aged and elderly people infected are greater than the young. Secondly, time series analysis for rabies cases and exposed cases to rabies confirmed that both time series have seasonal periodicity and are correlated with each other. The predictive rabies model shows that the rabies cases will be stable, whereas the exposed cases of rabies will increase in the next three years. Thirdly, panel regression showed that the rabies cases in Hubei province are comprehensively affected by population, GDP, air temperature and precipitation, and test results demonstrated statistically significant differences between low/high elevations and slopes. Finally, the limitations and the future research direction were further discussed.
Objective: Directed evolution of Escherichia coli phytase gene to obtain phytase mutants with improved thermostability. Methods: The mutant gene was obtained by error-prone PCR and the high-throughput screening methods of 96-well plate. After heterologous expression and purification, the mutant enzymes were characterized. Results: Three phytase mutants APPA1, APPA2 and APPA3 with significantly improved thermal stability were obtained through screening. The results of enzymatic properties analysis showed that the molecular weight of the three mutants was about 55kDa, and the optimum pH was 4.5, which was not significantly different from the wild-type. The thermal stability of mutants was significantly improved compared with the wild-type. Among them, the suitable temperature of APPA3 is 65℃, which is 5℃ higher than wild enzyme, and 50% of the enzyme activity is retained after treatment at 90℃ for 10 minutes. The three-dimensional structure simulation of the enzyme showed that the five mutation sites introduced new hydrogen bonds in the overall structure of the phytase. Conclusion: Obtaining Escherichia coli phytase mutants with improved thermal stability through directed evolution is of great significance to the industrial application of phytase and the study of the relationship between phytase structure and function.
Rifamycin SV has low toxicity, high curative effect, and broad antibacterial spectrum. It is mainly produced through the aerobic fermentation by Amycobacterium marinum, and oxygen supply limitation was always the critical factor that affects the rifamycin SV biosynthesis. In order to reduce the influence of oxygen limitation in the fermentation process and further increase the fermentation yield of rifamycin, an oxygen limitation directional model was established with anhydrous sodium sulfite, combining normal temperature and atmospheric pressure plasma mutagenesis(ARTP); a high-throughput screening method of rapid culture of the vinegar-producing strains in 24-well plates was proposed; the high-yielding rifamycin SV strain NSMXG-M126 that can tolerate hypoxia has been efficiently selected. The changes in the parameters of the fermentation metabolic state show that the high-yielding strain has better oxygen affinity. Under the same oxygen supply conditions, it showed a faster bacterial growth rate and rapid synthesis of rifamycin SV compared with the control. In the case of low oxygen supply, the fermentation unit reached 7 839mg/mL, which was 48% higher than the original strain. The results demonstrated that the mutant strains that tolerate hypoxia have higher production efficiency of rifamycin SV.
Background:Streptomyces spiramyceticus is used to produce spiramycin (SPM) in China. So far, the successful genetic modification has not been reported in S. spiramyceticus.Objective:The appropriate genetic transformation system of S. spiramyceticus was established in order to modify the composition of SPM and reduce the separation costs of SPM based on genetic operation.Methods:The conjugal transfer system was conducted by conjugation with Escherichia coli ET12567/pUZ8002, and the factors that influence the conjugation efficiency, including culture medium, antibiotic coverage time, and donor/recipient ratio, were investigated and optimized.Results:The experimental results showed that S. spiramyceticus spores were not suitable for conjugal transfer and the best medium for conjugal transfer was ISP4. The maximum transconjugants were obtained when the donor/recipient ratio was 103∶1. The best conjugation efficiency was 1.93×10-4 and the percentage of SPM changed significantly.Conclusion:The efficient and simple genetic transformation system for producing strain S. spiramyceticus was established. Based on this method, the sspA gene was knocked out and φC31 locus attB was integrated into S. spiramyceticus genome successfully, which laid the foundation for further biosynthetic gene modification in the strain.
Bacillus licheniformis 2709 is a strain of alkaline protease that has been put into industrial production due to its easy cultivation, GRAS status and perfect protein secretion ability. In order to improve the fermentation production performance of the strain, increase the utilization of culture medium components and the production of alkaline protease by the bacteria, the extracellular secretion enzyme system has been improved. With the help of homologous recombination mechanism, the xylanase gene (xynA) from Bacillus pumilus was introduced near the origin of replication and the lipase gene (lipY2) from Yarrowia lipolytica was introduced into the center of the origin of replication. The results showed that the activity of xylanase and lipase reached(58±2.07)U/mL and (207±10.62)U/mL, respectively when the strain was fermented in shake flask for 44h. Their efficient secretion and expression ability promoted the utilization rate of the fermentation substrate by Bacillus licheniformis, thus increasing the content of reducing sugar and total nitrogen in the medium and decomposing nitrogen compounds in the precipitate. Compared with the original strain, the bacterial biomass of the mutant strain was increased by 11.76%, the fermentation cycle of alkaline protease was shortened by 4h, and the yield of alkaline protease was increased by 14.41%. The enrichment of the secreted enzymes and the improvement of fermentation performance of Bacillus licheniformis 2709 provide a method for modification of Bacillus licheniformis as a microbial agent in the feed industry.
Objective:In order to obtain a lipase with high enzyme activity and high temperature resistance, the Thermomyces lanuginosus lipase was modified by rational design, which laid the foundation for the application of lipase in the feed industry, oil processing and biodiesel.Methods:A candidate site was found by phylogenetic analysis of the lid and loop regions of the typical domain of lipase. A lipase recombinant with significantly improved lipase activity and high temperature resistance was obtained through rational design and experimental verification, and a multi-copy vector was constructed to complete the evaluation of the enzyme production ability in a 50L fermentor.Results:The thermal stability of lipase was significantly improved after designing. The crude enzyme solution still retained 78.94% of the enzyme activity after being placed at 80℃ for 12h. After fermentation for 168h in a 50L fermentor, the enzyme activity of the supernatant reached 29 000U/mL.Conclusion:A novel high activity and high thermal stable lipase was successfully designed and obtained, which facilitates its large-scale production and industrial application.
The surface-enhanced Raman scattering (SERS) technology has received extensive attention in the field of pathogen detection due to its prominent advantages of high sensitivity, rapid detection, high specificity and non-destructive characterization.The research progress of marker-based SERS methods in the field of pathogen detection in recent years was summarized. Moreover, the construction and application of a multifunctional SERS detection platform for pathogenic bacteria were introduced. Finally, the future development of SERS as a real-time, efficient and reliable tool for detecting pathogens is prospected.
This article reviews the application and advances of bispecific nanobodies from the research progress of nanobodies and studies of bispecific nanobodies in treating infectious diseases, cancer, and immune system diseases. Moreover, it highlights the research hotspots and potential research areas at this current stage. For in-depth analysis, the differences between nanobodies and full-length monoclonal antibodies are compared and the unique advantages of bispecific nanobodies are demonstrated. The research progress of nanobodies has been summarized, while emphasizing the research hot spots of neutralizing nanobodies of the COVID-19, CAR-T cell therapy, and immune checkpoint therapy antibodies. In conclusion, a prospect analysis of the application prospects of bispecific nanobodies is carried out. It is pointed out that bispecific nanobodies are able to overcome the shortcomings of full-length antibody and become a form of the antibody with great druggability.
Rett syndrome (RTT) is a neurological disease caused by mutations in the X-linked gene methyl CpG binding protein 2 (Mecp2). RTT occurs mostly in girls, but the mechanism that Mecp2 mutations caused RTT symptoms is largely unknown. The recent studies of RTT mostly focus on the role that Mecp2 plays in the central nervous system. This article reviews the development history of RTT, the discovery and main effects of Mecp2, and then discusses the research progress of MeCP2 in lipid metabolism, mitochondrial metabolism, and autophagy. Overall, Mecp2 mutations can affect cholesterol and fat metabolism in patients and lead to body obesity and liver dysfunction; the morphology and function mitochondrial changes and the disorders of autophagy occur. Because of the above metabolic disorders, the quality of life of RTT patients is seriously reduced. These findings provide a theoretical basis for exploring the mechanism of RTT deeply and screening possible therapeutic targets.
Rheumatoid arthritis (RA) is a systemic, chronic autoimmune disease that is considered to be incurable. Less effective concentration of conventional drugs in the affected joints and more frequent adverse effects of even novel biological agents remain to be therapeutic challengs for RA. Advances in nanobiotechnology have facilitated the development of new classes of therapeutics with a focus on new drug delivery systems. Recent studies have shown that nanocarriers can significantly improve bioavailability of existing, traditional disease-modifying antirheumatic drugs (DMARS), and transdermal delivery can remarkably decrease toxic effects and adverse reaction of oral administration and injection of drugs. Here, nanomaterials used in the transdermal drug delivery system for RA therapy and target therapeutic strategies in the basic-research aspect of RA as well as the progress and existing issues of current nano-preparations are summarized so as to provide perspectives for further avenues of development and improved method of novel transdermal nanomaterial-loaded drug delivery systems.
As biological macromolecules to guide biological development and life function activities, DNA can also be used to construct nanomaterials. DNA hydrogels can be prepared with both DNA biological function and hydrogel characteristics and applied to the analysis and detection of environmental samples. Polymerase chain reaction, hybridization chain reaction, roll-ring amplification and DNA super-analysis self-assembly were compared and analyzed according to the method of preparing long DNA hydrogel chains. It is concluded that the single technology of DNA amplification cannot meet the experimental requirements, and the appropriate technology should be selected according to the experimental conditions. The synthesis principle and process of physical hydrogels and chemical hydrogels are introduced. Chemical gels are mainly based on DNA ligase or DNA terminal synthesis. The physical glue formation is mainly based on van der Waals force or hydrogen bond synthesis. Since physical hydrogels and chemical hydrogels are not sensitive to environmental changes, the synthesis process needs to be improved. By comparing and analyzing the characteristics of modification methods, the synthetic DNA hydrogel can respond quickly to environmental changes and be applied to environmental sample detection. In combination with the denaturation response characteristics of environmental sample concentration detection, the technical points and detection performance of colorimetric, fluorescence and electrochemical methods are analyzed. Colorimetric method uses color developing agent as a indicator signal. When the structure of hydrogel changes, the sample can be detected according to the color of the developing agent. Fluorescence analysis uses the color and intensity of fluorescence to detect the sample qualitatively or quantitatively. Electrochemical analysis uses electrical signals such as resistance and current to detect samples quickly. Compared with the large instrument analysis method, these methods have the characteristics of low detection limit, wide detection range, fast detection time and low sample cost, etc. It is a convenient and fast method with a wide application prospect. Finally, the performance and economy of detection are evaluated, and its application prospect is summarized and prospected.
The isothermal amplification technology of nucleic acid is a molecular amplification technology which can amplify nucleic acid efficiently in a constant temperature system. It can realize exponential growth of target gene in a short time. The microfluidic chip should not be too close to the surface, but should not be too close to the surface. Several steps, such as sample preparation, nucleic acid enrichment, purification and detection, should be integrated onto a “miniaturized”chip, and should be automatically treated to get the experimental result:“sample in, result out”. The combination of the isothermal nucleic acid amplification technology and microfluidic chip can not only realize rapid nucleic acid amplification, but also reduce the dependence on experimental equipment. It has a broad application prospect in bedside instant diagnosis and pathogen rapid screening.The principle of isothermal amplification technology and the application of isothermal amplification technology of nucleic acid based on microfluidic chip are reviewed, and the development trend and application prospect of integrated microfluidic chip are prospeced.
Human coagulation factor VII is a single chain glycoprotein that is vitamin K-dependent and plays an extremely important role in the process of coagulation,which has been widely used in the clinic. It can be used for hemostatic purposes such as hemophilia with inhibitors,congenital FVII deficiency, thrombocytopenia and traumatic bleeding caused by surgery or severe trauma. The gene recombination technology is an attractive methed which provides more effective ways to produce recombinant human coagulation factor VII on a large scale. In recent years, a variety of recombinant expression systems of human coagulation factor VII have been tried and established. In this paper, it is focused on the development and application of recombinant human coagulation factor VII in the expression systems of yeasts, mammalian cells and transgenic animals and compares the characteristics of post-translational modification of different mammalian cells, so as to provide reference for the further development of recombinant human coagulation factor VII in the recombinant expression system.
The prokaryotic expression protein system, especially Escherichia coli expression system, is a preferred method for obtaining exogenous proteins because of its advantages of simple operation, short cycle, high expression level and relatively low cost. However, one of the major factors to limit the extensive application of E.coli expression protein system is that the exogenous proteins, when expressed in a high amount, are often present in an insoluble form. With the understanding of protein folding dynamics, enzymes and molecular chaperones involved in protein folding, scientists have made great progress in promoting solubility of heterologous proteins by changing expression conditions, modifying host cells or using fusion tags. Here, we review the related progress in order to provide reference for related research.
With the intensification of coronavirus pandemic, the research and development of in vitro diagnostic technologies and products for SARS-CoV-2 attracts great attention in the global biomedical field. The research and development of in vitro diagnostic technologies and products is of great importance in enhancing the ability to prevent and control emerging infectious diseases. The global research status and future trend of SARS-CoV-2 diagnostic technologies and products was reviewed,including not only the traditional molecular diagnostic and immunodiagnostic technologies, but also the application of new technologies, such as CRISPR technology, biosensor technology, nanotechnology, in SARS-CoV-2 testing, and the research and development strategy and technical characteristics were also analyzed in order to provide a reference for the development of in vitro diagnostic technologies for emerging infectious diseases and the science and technology decision making of the government.
In recent years, with a growing number of approved ASO and siRNA drugs, RNA therapy has gradually moved from clinical to industrial application, and RNA therapy currently can be applied to the treatment of rare diseases, tumors, infectious diseases and others. On the whole, a certain foundation on domestic RNA therapy industry has been built,while few engaged institutions, weak research pipelines and lacking of key technologies are still the main reasons that limit the development of RNA therapy industry in China. The development trends of RNA therapy industry at home and abroad are combed and analyzed, and then some suggestions for the development of RNA therapy in China are put forward.
